Calcium induces electron transfer in NADPH oxidase 5

Abstract

Superoxide generated by non‐phagocyte NADPH oxidases (NOXs), such as NOX5 and DUOX, is of growing importance for vascular physiology and pathology. NOX5 and DUOX enzymes consist of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. They appear to be regulated by self‐contained Ca2+ binding domains (EFD). The basis for their catalytic profiles and regulation is unclear. Here, we demonstrated that Ca2+ gates heme reduction NOX5 in anaerobic conditions. To better understand this interaction, we monitored its Ca2+ and terbium (Tb3+) bindings by fluorescent spectroscopy using wild type and mutants of the recombinant EFD. The titration of EFD yielded a [Ca2+]0.5 of 4.5 μM, which is similar to published results, but the Ca2+ bindings appears to be tighter and occur in a cooperative manner. About 10 μM Ca2+ is required to fully saturate EFD and completely activate NOX5 superoxide production. EFD binds terbium with a [Tb3+]0.5 of 20 μM. The site‐directed labeling on Cys107 of EFD with IAEDNS had the same response to the Ca2+ level as the non‐labeled EFD. It reveals an anisotropy value of 0.11 and will be used to gather the structural information of the EFD‐flavoprotein complex using FRET. NOX5 contains a consensus peptide sequence that interacts with Ca2+‐bound calmodulin (CaM), and it has been demonstrated that this CaM/Ca2+ binding increases its sensitivity for free Ca2+. We used Troponin C (TnC)‐CaM chimeras to pinpoint the structural‐functional relationship to understand how individual EF‐hands motifs in CaM affect the electron transfer in NOX5. We are currently performing similar studies in DUOX. This work was supported by Beginner‐Grant‐in‐Aid from the American Heart Association